The present invention relates to the monitoring of cellular activity through the use of excitable markers. More particularly, the invention relates to a system and method for using a plurality of fluorescent probes to monitor cellular activity.
Presently, fluorescence microscopy is one of the most widely used microscopy techniques, as it enables the molecular composition of the structures being observed to be identified through the use of fluorescently-labeled probes of high chemical specificity, such as antibodies. However, its use is mainly confined to studies of fixed specimens because of the difficulties of introducing antibody complexes into living specimens. For proteins that can be extracted and purified in reasonable abundance, these difficulties can be circumvented by directly conjugating a fluorophore to a protein and introducing this back into a cell. It is believed that the fluorescent analogue behaves like the native protein and can therefore serve to reveal the distribution and behavior of this protein in the cell.
An exciting, new development in the use of fluorescent probes for biological studies has been the development of the use of naturally fluorescent proteins as fluorescent probes, such as green fluorescent protein (GFP). The gene for this protein has been cloned and can be transfected into other organisms. This can provide a very powerful tool for localizing regions in which a particular gene is expressed in an organism, or in identifying the location of a particular protein. The beauty of the GFP technique is that living, unstained samples can be observed. There are presently several variants of GFP which provide spectrally distinct emission colors.
Conventionally, fluorescence microscopy only worked well with very thin specimens or when a thick specimen was cut into sections, because structures above and below the plane of focus gave rise to interference in the form of out-of-focus flare. However, this can be overcome by optical sectioning techniques, such as multi-photon fluorescence microscopy.
Multi-photon fluorescence microscopy involves the illumination of a sample with a wavelength around twice the wavelength of the absorption peak of the fluorophore being used. For example, in the case of fluorescein which has an absorption peak around 500 nm, 900 nm excitation could be used. Essentially no excitation of the fluorophore will occur at this wavelength. However, if a high peak-power, pulsed laser is used (so that the mean power levels are moderate and do not damage the specimen), two-photon events will occur at the point of focus. At this point the photon density is sufficiently high that two photons can be absorbed by the fluorophore essentially simultaneously. This is equivalent to a single photon with an energy equal to the sum of the two that are absorbed. In this way, fluorophore excitation will only occur at the point of focus (where it is needed) thereby eliminating excitation of the out-of-focus fluorophore and achieving optical sectioning.
Often, multiple fluorophores are used, with each fluorophore having a different spectra, some of which may overlap. Typically, the ability to distinguish between the respective fluorophores is only possible where the excitation and emission spectra are separated, or where the fluorescence lifetimes are distinct.
Another approach is to selectively excite different fluorophores by using various excitation photon wavelengths, each of which will approximate the wavelength of the absorption peak of a corresponding fluorophore. Such an approach is not practical for a number of reasons. Firstly, it is difficult to rapidly tune the excitation wavelength of the laser providing the excitation photons. Secondly, there is typically a very broad excitation spectrum, so that such an approach makes it difficult to excite a single dye.
Thus, the need exists for a system and method for efficiently monitoring a plurality of fluorescent probes, and to selectively record the signal from those probes for subsequent analysis. The present invention addresses these needs.
Briefly, the present invention is directed to a system and method for monitoring cellular activity in a cellular specimen. According to one illustrative embodiment of the invention, a plurality of excitable markers are applied to the specimen. A multi-photon laser microscope is provided to excite a region of the specimen and cause fluorescence to be radiated from the region. The radiating fluorescence is processed by a spectral analyzer to separate the fluorescence into wavelength bands. The respective fluorescence bands are then collected by an array of detectors, with each detector receiving a corresponding one of the wavelength bands.
According to another embodiment, the invention is directed to a system for monitoring cellular activity in a cellular specimen that contains a plurality of excitable markers. The system includes a laser microscope that is operative to excite the markers in a region of the specimen, so that those markers in the region radiate fluorescence. The system also includes a tunable filter that is operative to process the fluorescence and to pass a portion of the fluorescence wavelengths radiated by the markers. The system still further includes a detector that is operative to receive the processed fluorescence wavelengths.
In still another embodiment, the invention is directed to a system for monitoring cellular activity, including a two-photon laser microscope that is operative to excite the markers in a region of the specimen such that the markers in the region radiate fluorescence. The system also includes a detector that is operative to receive non-descanned fluorescence from the specimen.